A continuing problem in the diagnosis of neurodegenerative diseases or conditions such as Alzheimer's has been to devise a reliable biomarker that provides a definitive indication of a physical pathology. Typically, a neurodegenerative disease is diagnosed based on behavior, signs of cognitive impairment, and various forms of radiological imaging, and a definitive diagnosis is obtained only upon autopsy.
Further, the cause of neurodegenerative diseases has been a mystery, making it difficult to know what biological factors to look for in terms of early warning signs that a neurodegenerative disease may be present. Post mortem analysis of brain tissue appears to implicate metal ions in neurodegenerative diseases. Proteins associated with these diseases bind metals as part of their normal function, but in neurodegenerative diseases, something causes the proteins to not fold around the metals properly, thereby revealing transition metal sites that can participate in oxidation-reduction reactions. This development leads to mild to severe amyloid angiopathy.
In neurodegenerative diseases such as Alzheimer's, Parkinson's Huntington's amyotrophic lateral sclerosis (ALS) and scrapie, oxidative modifications occur leading to pathological lesions. For example, tyrosine nitration is one of the earliest markers found in Alzheimer's disease brains and ALS. (Ischiropoulos, I. & Beckman, J S. “Oxidative stress and nitration in neurodegeneration: cause, effect, or association?” J. Clin. Invest. 2003; 111:163-69.) One of the most likely oxidants involved in nitrosylation of tyrosines in the central nervous system is derived from nitric oxide reacting with superoxide, called peroxynitrite. (Smith, M A, et al. “Widespread peroxynitrite-mediated damage in Alzheimer's disease.” J. Neuroscience. 1997; 17:2653-57)
The present inventor has previously reported the discovery that blood and other bodily fluids from normal individuals contain a significant number of antibodies, that, when treated with an oxidizing agent, become capable of binding self antigens. See, for example, the following publications:
McIntyre, J A. “The appearance and disappearance of antiphospholipid antibodies subsequent to oxidation-reduction reactions.” Thromb. Res. 2004; 114:579-87.
McIntyre, J A, Wagenknecht, D R, & Faulk, W P. “Autoantibodies unmasked by redox reactions.” J. Autoimmun 2005; 24:311-17.
McIntyre, J A, Wagenknecht, D R, & Faulk, W P. “Redox-reactive autoantibodies: Detection and physiological relevance. Autoimm. Rev. 2006; 5:76-83. and U.S Patent Application Publication No. 2005/0101016 A1.
The entire contents of these publications are incorporated herein by reference.
Such autoantibodies may be detected by treating the blood or other bodily fluid with an oxidizing agent and then using a screening assay to detect antibodies that bind a self antigen. It has been found that such autoantibodies are present in blood or other bodily fluids in a wide variety of isotypes and specificities. It has also been found that autoantibodies can be detected in a purified or fractionated immunoglobulin composition that has been treated with oxidizing conditions. Since the autoantibodies are not detected above a minimal baseline in blood or other bodily fluids from normal individuals or in immunoglobulin compositions pooled from normal individuals in the absence of an oxidation step, antibodies or autoantibodies having this property are referred to herein as “masked” antibodies or “masked” autoantibodies, and the process of treating blood or other bodily fluids or immunoglobulin preparations with oxidizing conditions is referred to herein as “unmasking” the masked antibodies or autoantibodies. Antibodies having the property of becoming masked or unmasked, depending on oxidation-reduction conditions may also be referred to herein as “redox antibodies”.
To date, masked autoantibodies that have been detected in the blood of normal individuals include the following:
TABLEMasked autoantibodies identified to date after redox conversion ofnormal plasma or IgG.Current list of redox-reactive autoantibodies*Antiphospholipid antibodies, aPS, aCL, aPE, aPC, LupusAnticoagulant (LA)Anti-glutamic acid decarboxylase (GAD)Anti-tyrosine phosphatase (IA-2)Anti-nuclear antibodies (ANA)Anti-cell organelles: nucleolus, lamin, Golgi, etc.Anti-granulocytes: neutrophils, monocytesAnti-B lymphocyteAnti-myeloperoxidaseAnti-tumor cells lines: Raji, Jurkat, U87MG, K562Anti-trophoblast and trophoblast basement membranes (TBM)Anti-factor VIIIAnti-PF4/heparin complexAnti-β2-glycoprotein IAnti-RBC (broad reactivity)*Additional specificities are anticipated upon further testing.Table abbreviations used:aCL, anticardiolipinaPC, antiphosphatidylcholineaPE, antiphosphatidylethanolamineaPS, antiphosphatidylserineAPPT, activated partial thromboplastin timedRVVT, dilute Russell's viper venom timeELISA, enzyme-linked immunosorbant assay
The present inventor has proposed that nitrosylation of tyrosine residues in and around the antibody hypervariable region may be a potential mechanism for antibody masking and unmasking. A change in nitration could produce conformational changes in an antibody binding site that result in alteration of the binding specificity of the antibody. To test this theory, hemin-treated and untreated samples of IgG were assayed for nitrated tyrosines and it was found that there was significant IgG nitrosylation after hemin exposure. See McIntyre, J. Autoimmun, cited above.
It is presumed that masked autoantibodies present in normal individuals do not cause harm to the normal individual, and may even play a yet unknown beneficial role. However, autoantibodies that become unmasked in the body, which can occur through physiological oxidative reactions, are believed to play a role in autoimmune diseases.
The present inventor has also reported the discovery of masked autoantibodies in samples of cerebral spinal fluid taken from normal individuals. See U.S. patent application Ser. No. 11/108,826; Sokol, D K, Wagenknecht, D R & Mcintyre, J A. “Testing for antiphospholipid antibody (aPL) specificities in retrospective “normal” cerebral spinal fluid (CSF)”. Clin. Develop. Immunol. 2004; 11:7-12. As with autoantibodies detected in the blood, the autoantibodies in cerebral spinal fluid from normal individuals can be detected in surprisingly large quantities by treating the cerebral spinal fluid sample with oxidizing conditions, such as with an oxidizing agent or the use of electromotive force and then using a screening assay to detect antibodies that bind self antigens. Such autoantibodies are not detected above a minimal baseline in the cerebral spinal fluid taken from a normal individual that is not subjected to oxidizing conditions. Here again, it can be presumed that autoantibodies that may be present in cerebral spinal fluid of a normal individual in their masked form do not cause harm to the individual, and may play a yet unknown beneficial role; however, it is apparent that the autoantibodies could cause damage if they were to become unmasked in the cerebral spinal fluid. These results suggested that autoantibodies may be involved in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, and that these diseases could be triggered or aggravated by unmasking of masked autoantibodies in the cerebral spinal fluid. This theory is supported by the discovery, discussed above, that metal ions are implicated in neurodegenerative diseases. For example, if transition metal sites are exposed by protein misfolding, such exposed sites could promote oxidation-reduction reactions that lead to unmasking of autoantibodies. Unmasked antibodies such as antiphospholipid autoantibodies can interact with phospholipids and phospholipid-binding proteins in brain cells and may therefore cause many of the lesions and shrinkage of the brain that are seen in MRI studies of the Alzheimer patient brains and can cause the physical damage seen in other types of neurodegenerative diseases.
As reported herein, it has now been discovered that autoantibodies are not detected in the post mortem cerebral spinal fluid of Alzheimer's patients subsequent to oxidizing reactions. In contrast, post mortem control cerebral spinal fluid samples from patients with no history of neurodegenerative diseases do possess autoantibodies subsequent to oxidation reactions. These results suggest that certain neurodegenerative diseases or conditions can be characterized by detecting the presence of active or unmasked autoantibodies in cerebral spinal fluid. Moreover, as further discussed herein, it has been discovered that cerebral spinal fluid of confirmed Alzheimer patients that is treated with an oxidizing agent such as hemin does not show a dramatic increase in the amount of detectable autoantibodies, as compared with untreated cerebral spinal fluids, which indicate that an unmasking process has occurred in a diseased subject, such that the level of masked autoantibodies becomes depleted. These results alternatively suggest that the presence of a neurodegenerative disease or condition can be detected by comparing the amount of autoantibodies in a sample of cerebral spinal fluid that is untreated with the sample of cerebral spinal fluid that is treated with an oxidizing agent such as hemin or electromotive force.
In addition to the discovery that Alzheimer's post mortem cerebral spinal fluid lacks redox-reactive autoantibodies, it has been shown that the autoantibodies unmasked in cerebral spinal fluid from a normal individual can stimulate signal transduction reactions when assayed using a mouse synaptosome model. This finding may relate to the brain pathology observed in neurodegenerative diseases at autopsy since the unmasked autoantibodies from an individual have been shown to phosphorylate the extracellular signal regulated kinase (ERK1/2), a member of the mitogen activated protein kinase (MAPK) cascade. Such phosphorylation reactivity either in the cytosol and/or the nucleus can promote gene expression leading to proliferation, transformation, and differentiation or programmed cell death (apoptosis). Related phosphorylation pathways, for example, JNK and p38 also would be expected to participate. Apoptosis of neurons as well as interference with memory and motor functions in the brain subsequent to ERK1/2 phosphorylation are known responses resulting from activation of this stimulation pathway. (For review, references to ERK1/2 phosphorylation outcomes are found in: Adams, JP and Sweatt, J D. “Molecular Psychology: Roles for the ERK MAP Kinase Cascade in Memory”. Annu. Rev. Pharmacol. Toxicol. 2002; 42:135-63; Hindley, A, and in, Kolch, W. “Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases”. J. Cell Science, 2002; 115:1575-81 and in, Cheung, ECC and Slack, R S. “Emerging Role for ERK as a Key Regulator of Neuronal Apoptosis”. Science, 2004; 251:1-3). A direct pathogenic role for antiphospholipid antibodies has also been shown in: Chapman, J, et al. “Antiphospholipid antibodies permeabilize and depolarize brain synaptosomes”. Lupus 1999; 8:127-33.
It is proposed that the failure to find redox-reactive autoantibodies in Alzheimer's disease cerebral spinal fluid is due to their depletion caused by disease-associated nitrosylation of proteins that are characteristic of certain neurodegenerative diseases. The autoantibodies are not detected because they have targeted and are bound to the neurons in the diseased brain. Recent evidence for antibody deposition in the brain cells can be found in: DeAndrea, M R. “Evidence that immunoglobulin-positive neurons in Alzheimer's disease are dying via the classical antibody-dependent complement pathway”. Am J Alzheimer's Dis Other Dimentias. 2005; 20:144-50. Moreover, chronic activation of ERK1/2 is supported by failure to detect redox-reactive autoantibodies in Alzheimer's cerebral spinal fluid subsequent to oxidation. That this can lead to neurodegenerative diseases was reported by: Colucci-D'Amato L, et al. “Chronic activation of ERK and neurodegenerative diseases”. Bioassays, 2003; 25:1085-95.